Normal cell development requires the interaction of RNA polymerase with many factors that regulate transcriptional activity. We study transcription from bacteriophage T4 middle promoters in which the initiation of transcription by E. coli RNA polymerase requires the phage-encoded transcriptional activator, MotA protein, and the T4 co- activator, AsiA protein. This system is a simple model for examining how factors can change the specificity of a polymerase.T4 middle promoters contain a -10 region that is recognized by the sigma-70 subunit of RNA polymerase and a MotA box sequence centered at -30 that is bound by MotA. Previously, we have determined an apparent dissociation constant of 130 nM for MotA binding to a MotA box. We have extended this study by investigating how the loss or modification of base determinants within the MotA box sequence 5TTTGCTTTA3 (positions - 34 to -26 of a middle promoter) affects MotA function. Changing the C:G bp at position -30 to either an A:T, G:C, or T:A resulted in a MotA box that was bound by MotA and was active for MotA/AsiA dependent transcription in vitro. The 5-methyl residue on the thymine at position -29, a major groove contact, contributed to MotA binding, decreasing the apparent dissociation constant by a factor of 4. In contrast, converting the T:A at -32 to a C:I bp, a change that affects the major but not the minor groove, yielded a MotA box that was bound similarly to wild type. Furthermore, even replacing the T:A bps at positions -34 through -32 with C:I bps only modestly impaired binding. Taken together, these results suggest that MotA uses minor groove contacts upstream and major groove contacts downstream of the center GC and does not require any specific base feature at the C:G bp at position -30. However, our methylation interference analyses indicated that neither the binding of MotA nor the binding of polymerase/MotA/AsiA to the middle promoter PuvsX was inhibited by premethylation of guanines and adenines, suggesting that minor groove binding upstream of position -30 does not require contact with any specific T:A bp. AsiA is a protein of 90 amino acids that binds tightly to the sigma-70 subunit of RNA polymerase. AsiA inhibits transcription from host promoters and acts as a co-activator of transcription from T4 middle promoters in the presence of MotA. The C-terminal portion of AsiA contains an unusually high number of hydrophobic amino acids, with phenylalanines at positions 73 and 77 and tyrosines at positions 81, 83, and 87. Because protein domains that are involved in protein-protein or protein-DNA interactions can have this type of amino acid sequence, we targeted the C-terminal region of AsiA for mutagenesis. We constructed an AsiA mutant in which the tyrosines at amino acids 81 and 83 were changed to alanines (ARA) and AsiA mutants with C-terminal deletions of 4 aa, 12 aa, or 17 aa. Wild type AsiA is extremely toxic when expressed in E. coli, presumably because of its tight binding to sigma-70. The AsiA proteins with deletions of 4 and 12 amino acids and the ARA protein were also toxic when expressed in E. coli. These mutant AsiA proteins also complemented a T4 asiA amber mutant phage for growth under nonsuppressing conditions. In contrast, the protein with the deletion of 17 amino acids was less toxic, and it did not complement the mutant phage. However, using purified proteins, we found that each of the mutant AsiA proteins bound to sigma-70 and functioned as a co-activator in vitro. Our results suggest that while the C-terminal 17 amino acids of AsiA are not required for function in a purified in vitro system, these amino acids may contribute to the function or stability of the AsiA protein in vivo. - transcription, bacteriophage, activation